Inverse Regulation of the Nuclear Factor-κB Binding to the p53 and Interleukin-8 κB Response Elements in Lesional Psoriatic Skin  Claus Johansen, Esben Flindt, Knud Kragballe, Jeanette Henningsen, Majken Westergaard, Karsten Kristiansen, Lars Iversen  Journal of Investigative Dermatology  Volume 124, Issue 6, Pages 1284-1292 (June 2005) DOI: 10.1111/j.0022-202X.2005.23749.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Nuclear factor-κB (NF-κB) DNA binding activity in lesional and non-lesional psoriatic skin. Keratome biopsies were taken from patients with psoriasis vulgaris. The biopsies were homogenized and the nuclear fraction isolated and incubated with a labelled oligonucleotide identical to either the κB site from the p53 promoter (a), the interleukin-8 (IL-8) promoter (b), or the major histocompatibility complex class I (MHC-I) promoter (c). The NF-κB binding activity was then determined by electrophoretic mobility shift assay. Journal of Investigative Dermatology 2005 124, 1284-1292DOI: (10.1111/j.0022-202X.2005.23749.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Characterization of the nuclear factor-κB (NF-κB) binding complex. Supershift analysis of the NF-κB dimer in lesional and non-lesional psoriatic skin was carried out. Antibodies directed against the indicated NF-κB/Rel proteins were added to the incubation mixture 10 min prior to the addition of the labelled oligonucleotide identical to either the p53 κB site (a) or the interleukin-8 (IL-8) κB site (b). Cultured human keratinocytes were stimulated with IL-1β (10 ng per mL) for 15 min before the nuclear fraction was isolated and analyzed by electrophoretic mobility shift assay (c). Antibodies directed against p65 and p50 were added to the incubation mixture 10 min prior to the addition of the labelled oligonucleotide identical to the p53 κB site. Journal of Investigative Dermatology 2005 124, 1284-1292DOI: (10.1111/j.0022-202X.2005.23749.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Nuclear factor-κB (NF-κB) DNA binding activity in lesional psoriatic skin treated with calcipotriol. Nuclear extracts were isolated from lesional psoriatic skin treated with either calcipotriol or vehicle for 4 d. The NF-κB DNA binding activity to the p53 κB site (a) and the interleukin-8 (IL-8) κB site (b) were then assayed by electrophoretic mobility shift assay. Journal of Investigative Dermatology 2005 124, 1284-1292DOI: (10.1111/j.0022-202X.2005.23749.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 p53 and interleukin-8 (IL-8) expression in psoriatic skin. (a) Real-time RT-PCR was carried out on RNA from six paired biopsies from lesional and non-lesional psoriatic skin. Numbers above 1 indicate an increased expression, whereas numbers below 1 indicate a decreased expression in lesional compared with non-lesional psoriatic skin. (b, c) Whole-cell extracts from psoriatic skin biopsies were isolated and then the IL-8 (b) and p53 (c) protein level was determined by western blotting using antibodies directed against either the IL-8 or the p53 protein. Equal loading was confirmed by incubation with an anti-transcription factor II B (TFIIB) or anti-β-actin antibody. Journal of Investigative Dermatology 2005 124, 1284-1292DOI: (10.1111/j.0022-202X.2005.23749.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Expression of the nuclear factor-κB (NF-κB)/Rel subunits in lesional psoriatic skin. (a) The mRNA expression levels of the NF-κB/Rel subunits in lesional and non-lesional psoriatic skin were determined by real-time RT-PCR. Numbers above 1 indicate increased expression in lesional compared with non-lesional psoriatic skin. (b) Whole-cell protein extracts were prepared from lesional and non-lesional psoriatic skin, and separated by SDS-PAGE on an 8%–16% gradient gel. After electroblotting, the separated proteins were probed with anti-p50, anti-p65, and anti-RelB antibodies. Equal loading was confirmed by incubation with an anti-TFIIB antibody. Journal of Investigative Dermatology 2005 124, 1284-1292DOI: (10.1111/j.0022-202X.2005.23749.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 × 100 LSM micrographs of human non-lesional and lesional psoriatic skin biopsies. Paired samples from involved (+Inv) and non-involved (-Inv) psoriatic skin are presented. Insets show details blown up from × 250 LSM micrographs. Red (SYTOX DNA stain) demonstrates the location of nuclei, green (Alexa488-conjugated antibody) demonstrates the location of nuclear factor-κB (NF-κB), and yellow indicates co-localization. These results were conducted on biopsies from three different patients. Journal of Investigative Dermatology 2005 124, 1284-1292DOI: (10.1111/j.0022-202X.2005.23749.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Analysis of IκB kinase (IKK)α/β/γ, IκBα, and IκBβ in psoriatic skin. (a, b, c) Whole-cell extracts were prepared from lesional and non-lesional psoriatic skin. The proteins were separated on an 8%–16% gradient gel and blotted on a nitrocellulose membrane. After blotting, the membrane was probed with the indicated antibodies. Equal loading was confirmed by incubation with an anti-TFIIB antibody. Journal of Investigative Dermatology 2005 124, 1284-1292DOI: (10.1111/j.0022-202X.2005.23749.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions